Plant defensive responses to insect eggs are inducible by general egg-associated elicitors

Egg deposition by herbivorous insects is well known to elicit defensive plant responses. Our study aimed to elucidate the insect and plant species specificity of these responses. To study the insect species specificity, we treated Arabidopsis thaliana with egg extracts and egg-associated secretions of a sawfly (Diprion pini), a beetle (Xanthogaleruca luteola) and a butterfly (Pieris brassicae). All egg extracts elicited salicylic acid (SA) accumulation in the plant, and all secretions induced expression of plant genes known to be responsive to the butterfly eggs, among them Pathogenesis-Related (PR) genes. All secretions contained phosphatidylcholine derivatives, known elicitors of SA accumulation and PR gene expression in Arabidopsis. The sawfly egg extract did not induce plant camalexin levels, while the other extracts did. Our studies on the plant species specificity revealed that Solanum dulcamara and Ulmus minor responded with SA accumulation and cell death to P. brassicae eggs, i.e. responses also known for A. thaliana. However, the butterfly eggs induced neoplasms only in S. dulcamara. Our results provide evidence for general, phosphatidylcholine-based, egg-associated elicitors of plant responses and for conserved plant core responses to eggs, but also point to plant and insect species-specific traits in plant–insect egg interactions.


Supplementary Tables
Statistical details on phytohormone and camalexin data presented in Fig. 1.   other, which may be assigned to the molecular formula C44H77NO8P (e.g., [M+H] + of PC 36:6) due to high mass accuracy (Δm/z less than 1.6 ppm).This is supported by the fact that isomer 1 could be identified as 18:3/18:3 PC by means of a reference standard.(b) Isomer 1 and 2 exhibit three MS/MS fragmentations characteristic for a phosphocholine group in the molecule.This observation further narrows the possible lipid class for isomer 2, basically to PCs and sphingomyelins.(c) A Lipid Maps® search (https://www.lipidmaps.org/)yielded a total of 17 possible structures with the listed search criteria for the specific exact mass-to-charge ratio of isomer 2 (m/z 778.5393).One of these is PC 18:3(9Z,12Z,15Z)/18:3(9Z,12Z,15Z), which could already be assigned to isomer 1 using a reference standard.

Method: Extraction and analysis of salicylic acid and camalexin from leaves
For quantification of salicylic acid and camalexin concentrations in A. thaliana leaves, the leaf material was homogenized in 2 ml tubes with beats (Zirconox,Mühlmeier Mahltechnik,Bärnau,Germany) in the FastPrep®-24 instrument (MP Biomedicals, Solon, USA) at 6 m s -1 for 20 s.We added 500 to 1000 µl ethyl acetate and 2 µl d4-SA internal standard (10 ng µl -1 ) (OlChemIm Ltd., Olomouc, Czech Republic).Samples were again homogenized in ethyl acetate 2 x 20 s at 6 m s -1 .Samples were centrifuged for 10 min at 4°C and 13.000 rpm using an Eppendorf® centrifuge 5427R with rotor Hamburg,Germany).The extraction was repeated with pure ethyl acetate.The supernatants of the two extraction steps were combined.After vaporization of the supernatant with the Eppendorf Concentrator 5301 vacuum concentrator, SA and camalexin were re-eluted in 300 µl 70% methanol with 0.1% formic acid (v/v) under 10 min vortexing at room temperature.The samples were centrifuged for 15 min at 13,000 rpm and room temperature.Finally, 200 µl of the supernatant were transferred to glass vials.

Method: Analysis of phosphatidylcholines in egg-associated secretions
Egg-associated secretions of the three insect species studied were subjected to chromatographic lipid separations for both untargeted QTOF and targeted QQQ analysis.
The separations were conducted on a Poroshell 120 EC-C8 column (3.0 x 150 mm, 2.7 μm; Agilent Technologies).A mobile phase system consisting of water (solvent A) and acetonitrile/methanol (1:1, v:v; solvent B), both acidified with 0.1% formic acid, was used for gradient elution at an initial composition of 40:60 (A:B, v:v) and a flow rate of 0.5 ml min -1 .The column was kept at 30°C, and the injection volume was 10 µl.
QTOF analysis was performed in scan mode in the range of m/z 50-1,500 with a scan rate of 2 spectra.
The reference masses m/z 121.0509 and m/z 922.0098 were continuously supplied by an isocratic pump.

Figure S2 .
Figure S2.Identification of the 36:6 PC isomer 2 found in oviduct secretion of Diprion pini (Dp).(a) HPLC coupled to high-resolution mass spectrometry showed two signals eluting close to eachother, which may be assigned to the molecular formula C44H77NO8P (e.g., [M+H] + of PC 36:6) due to high mass accuracy (Δm/z less than 1.6 ppm).This is supported by the fact that isomer 1 could be identified as 18:3/18:3 PC by means of a reference standard.(b) Isomer 1 and 2 exhibit three MS/MS fragmentations characteristic for a phosphocholine group in the molecule.This observation further narrows the possible lipid class for isomer 2, basically to PCs and sphingomyelins.(c) A Lipid Maps® search (https://www.lipidmaps.org/)yielded a total of 17 possible structures with the listed search criteria for the specific exact mass-to-charge ratio of isomer 2 (m/z 778.5393).One of these is PC 18:3(9Z,12Z,15Z)/18:3(9Z,12Z,15Z), which could already be assigned to isomer 1 using a reference standard.

Table S2 .
Statistical details on gene expression data presented in Fig.2.The data were statistically evaluated with ANOVA and Tukey post hoc test.

Table S3 .
Statistical details on phosphatidylcholine data in Fig.3.

Table S4 .
Statistical details on salicylic acid data presented in Fig.4.

Table S5 .
Statistical details on salicylic acid data presented in Fig.5.

Table S6 .
Analyzed genes and qRT-primers designed by PerlPrimer (Marshall 2004) and obtained from Eurofins Genomics.